Nitric oxide regulates interactions of PMN with human brain microvessel endothelial cells

https://doi.org/10.1016/j.bbrc.2004.08.062Get rights and content

Abstract

The hypothesis that the NO/cGMP pathway modulates PMN adhesion to human brain microvessel endothelial cells (HBMEC) was examined. Human PMN were incubated with resting or TNF-α-treated endothelial monolayers, and adhesion was quantified by light microscopy. TNF-α upregulated PMN adhesion in a time-dependent manner. Treatment of HBMEC with the NO donors SNP and DETA NONOate for 4 or 24 h decreased PMN adhesion. This was completely reversed by the guanylyl cyclase inhibitor ODQ, while addition of a cGMP agonist (8-Br-cGMP) decreased PMN adhesion. NO donors did not affect the levels of E-selectin or ICAM-1 in HBMEC. However, pre-treatment of PMN with NO donors or 8-Br-cGMP decreased their adhesion to recombinant E-selectin and ICAM-1, suggesting an effect of NO on PMN. These findings indicate that NO modulates PMN–HBMEC interactions through cGMP and decreases the binding of PMN to the adhesion molecules E-selectin and ICAM-1.

Section snippets

Materials and methods

Human brain microvessel endothelial cells. Primary cultures of human brain microvessel endothelial cells (HBMEC) were established from brains at autopsy as previously described [17]. The endothelial nature of these cells was confirmed by the positive staining for Factor VIII related antigen and binding of Ulex europeaus agglutinin. Cells were grown on fibronectin coated 96-well plates and cultured in Medium 199 with 10% horse serum. Nine- to ten-day-old confluent cultures were used. Several

Results

HBMEC were first treated with TNF-α, with or without the various donors and inhibitors for 4 or 24 h; 4 h for maximal upregulation of E-selectin and 24 h for ICAM-1. PMN were then placed on top of the monolayers and allowed to adhere for 20 min. After fixation and staining, the amount of adhesion was quantified by counting the number of PMN bound in 4 peripheral and 1 central field under the microscope. TNF-α treatment increased adhesion by threefold after 4 h and fivefold after 24 h. Adhesion was

Discussion

The blood–brain barrier (BBB), formed by the endothelial cells of the cerebral microvessels, regulates what enters the brain. Adhesion and activation of circulating leukocytes is an important aspect of the inflammatory response. The BBB normally prevents the movement of leukocytes into the brain. In acute cerebral inflammatory reactions, PMN infiltrate the brain. We have previously shown that human brain microvessel endothelial cells (HBMEC) express the adhesion molecules E-selectin and ICAM-1

Acknowledgments

This study was supported by grants from the Canadian Institutes of Health Research, the Canadian Stroke Network, and the Multiple Sclerosis Society of Canada. D. Wong was the recipient of a Multiple Sclerosis Society of Canada postdoctoral fellowship. We thank Dr. Kakuri Omari, Chris Bladen, and Dorota Kwasnicka for their expert technical assistance.

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